Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen–induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures - Blumbach - 2009 - Arthritis amp; Rheumatism - Wiley Online LibraryArthritis RheumatismVolume 60, Issue 12 p. 3676-3685 Cartilage Biology Free Access Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen–induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures K. Blumbach, Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, Germany Drs. Blumbach and Bastiaansen-Jenniskens contributed equally to this work.Search for more papers by this authorY. M. Bastiaansen-Jenniskens, Department of Orthopaedics, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, and TNO Quality of Life, Leiden, The Netherlands Drs. Blumbach and Bastiaansen-Jenniskens contributed equally to this work.Search for more papers by this authorJ. DeGroot, Business Unit BioSciences, TNO Quality of Life, Leiden, The NetherlandsSearch for more papers by this authorM. Paulsson, Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, GermanySearch for more papers by this authorG. J. V. M. van Osch, Department of Orthopaedics and Department of Otorhinolaryngology, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, The NetherlandsSearch for more papers by this authorF. Zaucke, Corresponding Author frank.zaucke@uni-koeln.de Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, GermanyCenter for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, GermanySearch for more papers by this author K. Blumbach, Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, Germany Drs. Blumbach and Bastiaansen-Jenniskens contributed equally to this work.Search for more papers by this authorY. M. Bastiaansen-Jenniskens, Department of Orthopaedics, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, and TNO Quality of Life, Leiden, The Netherlands Drs. Blumbach and Bastiaansen-Jenniskens contributed equally to this work.Search for more papers by this authorJ. DeGroot, Business Unit BioSciences, TNO Quality of Life, Leiden, The NetherlandsSearch for more papers by this authorM. Paulsson, Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, GermanySearch for more papers by this authorG. J. V. M. van Osch, Department of Orthopaedics and Department of Otorhinolaryngology, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, The NetherlandsSearch for more papers by this authorF. Zaucke, Corresponding Author frank.zaucke@uni-koeln.de Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, GermanyCenter for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, GermanySearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Objective Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril–associated proteins type IX collagen and cartilage oligomeric matrix protein (COMP) during cartilage matrix formation. Methods Primary chondrocytes from mice deficient in type IX collagen and COMP (double-deficient) were cultured in monolayer or alginate beads. Anchorage of matrix proteins, proteoglycan and collagen content, collagen crosslinks, matrix metalloproteinase activity, and mechanical properties of the matrix were measured. Electron microscopy was used to study the formation of fibrillar structures. Results In cartilage lacking both type IX collagen and COMP, matrilin 3 showed decreased matrix anchorage. Less matrilin 3 was deposited in the matrix of double-deficient chondrocytes, while larger amounts were secreted into the medium. Proteoglycans were less well retained in the matrix formed in alginate cultures, while collagen deposition was not significantly affected. Electron microscopy revealed similar cartilage collagen fibril diameters in the cultures of double-deficient and wild-type chondrocytes. In contrast, a larger fibril diameter was observed in the matrix of chondrocytes deficient in only type IX collagen. Conclusion Our results show that type IX collagen and COMP are involved in matrix assembly by mediating the anchorage and regulating the distribution of other matrix macromolecules such as proteoglycans and matrilins and have counteracting effects on collagen fibril growth. Loss of type IX collagen and COMP leads to matrix aberrations that may make cartilage more susceptible to degeneration. The cartilage extracellular matrix (ECM) is a complex alloy of proteins and proteoglycans, the composition of which defines tissue structure and physiologic functions (1). Changes in matrix composition are likely to result in impairment of matrix integrity and lead to an increased susceptibility for cartilage degeneration. Human genetics studies have indeed shown that mutations in matrix proteins can predispose for disease such as chondrodysplasia and/or osteoarthritis (OA) (2, 3). The role of many of the matrix proteins is still uncertain, and it is often not known how deficiencies in specific proteins predispose for cartilage degeneration. Cartilage collagen fibrils provide stiffness to the cartilage matrix. Proteoglycans, mainly aggrecan, account for the osmotic pressure and are therefore important to maintain the high water content of cartilage (4). Type IX collagen, in combination with type II collagen and type XI collagen, is a key component in the cartilage collagen fibril (5) and is thought to limit its lateral growth by binding to the fibril surface (6, 7). Type IX collagen can also act as a molecular bridge between fibrillar collagens and other ECM components, such as matrilin 3 (7) and cartilage oligomeric matrix protein (COMP; thrombospondin 5) (8, 9). COMP in turn interacts with matrilins and aggrecan as well as with type I collagen and type II collagen and is known to influence type I and type II collagen fibrillogenesis (10). Several mouse knockout models have been generated to gain further insight into the role of single ECM proteins, including type IX collagen and COMP, in cartilage matrix assembly and function. COMP-deficient mice lack an obvious phenotype (11), possibly due to functional compensation by other matrix proteins. Mice homozygous for the inactivated Col9a1 allele display degenerative changes in articular cartilage, which are first detected at the age of 6 months (12, 13). These mice have cartilage collagen fibrils with a larger diameter than wild-type (WT) (7). They also show alterations in the expression of matrix metalloproteinases (MMPs) and in mechanical properties, similar to what is seen during the development of human OA (14, 15). The loss of proteoglycans and adapter proteins (e.g., matrilin 3), which mediate interactions between collagen fibrils and their environment, has been suggested to be the molecular basis for the altered biomechanical properties (7, 15, 16). It has been unclear whether the loss of both type IX collagen and COMP enhances the matrix-assembly phenotype observed in type IX collagen–deficient animals, and whether type IX collagen and COMP play compensatory or antagonistic roles during matrix assembly. Recently, we and other investigators established mouse lines deficient in type IX collagen and COMP or in type IX collagen and multiple thrombospondins (17, 18). In these mice, disorganized growth plate architecture and alterations in matrix deposition were observed both at birth and at later stages of development (17). Loss of other thrombospondins in addition to COMP results in an even more pronounced phenotype. When subjected to running exercise, both single- and double-deficient mice showed flattening of the articular surface and an increased susceptibility for OA. However, fibrillation of the surface was seen only in mice lacking both type IX collagen and COMP (18). To date, the molecular events leading to these phenotypes are not well understood. In this study, we set out to analyze the contribution of collagen fibril–associated proteins to matrix assembly, by using our previously described mouse model lacking both type IX collagen and COMP and culturing primary chondrocytes derived from these mice in both monolayer and in an alginate-based 3-dimensional (3-D) culture system. Analyses of proteoglycan and collagen expression as well as matrix deposition, together with the study of the fibrillar structures formed, provided further insight into the functional interaction of type IX collagen and COMP. Type IX collagen–deficient mice (12) were kindly provided by the Peter Bruckner group (Muenster, Germany) and were bred onto a C57BL/6 background for at least 5 generations. To generate mice deficient in both type IX collagen and COMP (17), type IX collagen–deficient mice were intercrossed with mice lacking COMP (11), kindly provided by Åke Oldberg (Lund, Sweden), on a C57BL/6 background. C57BL/6 mice were used as WT controls. For immunohistologic stainings, the following primary antibodies were used: a mouse monoclonal antibody directed against human type II collagen (1:1,000 dilution; Calbiochem, La Jolla, CA), an affinity-purified rabbit polyclonal antibody against the NC4 domain of mouse type IX collagen (1:2,000 dilution) (7), a rabbit polyclonal antibody against bovine COMP (1:1,000 dilution) (19), and an affinity-purified rabbit polyclonal antibody against mouse matrilin 3 (1:1,000 dilution) (20). As secondary antibodies, Alexa Fluor 488–conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG (both from Molecular Probes, Leiden, The Netherlands) were applied. For histochemical and immunohistochemical stainings, 4% paraformaldehyde–fixed, paraffin-embedded tissue sections (5 μm) were dewaxed in xylol and rehydrated in isopropanol, 96%, 70%, and 50% ethanol, and water. To analyze the general tissue morphology and proteoglycan deposition, combined hematoxylin–eosin–Alcian blue staining (0.015% in 80% ethanol and 20% glacial acetic acid; 20 minutes) was performed. For immunohistologic staining, sections were digested with testicular hyaluronidase (10 mg/ml) for 30 minutes at 37°C, followed by permeabilization with 0.1% Triton X-100 and blocking in 10% fetal calf serum. Cultured monolayer cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes, permeabilized with 0.1% Triton X-100, and blocked with 2% normal goat serum. Both sections and cells were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hour at room temperature. The rib cages of double-deficient and WT mice ( 1 week old) were isolated, freed from surrounding noncartilaginous tissue, and frozen at −80°C. Proteins were extracted as described previously (21). For the detection of matrilin 3 by immunoblotting, samples were electrophoresed on 8% sodium dodecyl sulfate–polyacrylamide gels, electrotransferred onto nitrocellulose membranes, blocked with 5% skim milk, and incubated overnight at 4°C with an affinity-purified rabbit polyclonal antibody directed against matrilin 3 (1:1,000 dilution) (20). After washing, the membrane was incubated with horseradish peroxidase–conjugated donkey anti-rabbit IgG (1:2,000 dilution; Amersham Biosciences, Roosendaal, The Netherlands) for 1 hour at room temperature. Antibody detection was performed using 2.5 mM luminol, 0.4 mM p-coumaric acid, 0.01% H2O2 as a luminescent agent (Fluka, Buchs, Switzerland), and exposure on x-ray films. Primary chondrocytes were isolated from the rib cages and cultured in monolayer and in alginate beads, as described previously (7, 22). The medium was changed 3 times weekly and was stored for further analysis. Alginate beads were harvested and analyzed after 3 weeks of culture. Alginate beads were dissolved by adding 75 μl per bead of 55 mM sodium citrate and 20 mM EDTA in 150 mM NaCl for 20 minutes at room temperature. The suspension was centrifuged for 10 minutes at 1,000 revolutions per minute (Eppendorf, Hamburg, Germany) to separate the cells surrounded by the cell-associated matrix (the pellet) from components originating predominantly from the \"interterritorial” or further-removed matrix (the supernatant), as described previously (23-25). Alginate beads or samples of separated cell-associated matrix and further-removed matrix were digested overnight at 56°C with papain (200 μg/ml papain in 50 mM EDTA, pH 5.3, and 5 mM L-cysteine). Glycosaminoglycans were quantified using the dimethylmethylene blue (DMMB) assay (26). The metachromatic reaction with DMMB was monitored with a spectrophotometer, and the ratio A540:A595 was used to determine the glycosaminoglycan amount, with chondroitin sulfate C (Sigma, Munich, Germany) as standard. The amount of DNA in each papain-digested sample was determined using ethidium bromide with calf thymus DNA (Sigma) as standard. For high-performance liquid chromatography of amino acids (hydroxyproline) and collagen crosslinks (hydroxylysylpyridinoline and lysylpyridinoline), the samples were processed as described previously (22) and analyzed by reverse-phase high-performance liquid chromatography, using the methods described by Beekman et al (25) and Bank et al (27). The quantities of crosslinks were expressed as the number of residues per collagen molecule, assuming 300 hydroxyproline residues per triple helix. To determine total MMP activity, the culture medium was analyzed as described previously (23), using 5 μM fluorogenic substrate TNO211-F (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Cys[Fluorescein]-Ala-Lys-NH2). Because the alginate beads were constantly in culture medium, the medium represents the MMP activity in the alginate bead. The MMP activity in each sample was calculated as the difference in the initial rate of substrate conversion (linear increase in fluorescence in time, expressed as relative fluorescence units per second) between samples with and those without the addition of BB94. Fluorescence was measured for 6 hours at 30°C, using a CytoFluor 4000 reader (Applied Biosystems, Foster City, CA). For mechanical characterization, 4 × 106 cells/ml in 1.2% (weight/volume) alginate disks, 3 mm thick and 6 mm in diameter, were used. The disks were prepared and tested as previously described (22, 28). To ensure that enough matrix was produced and to test its functionality, disks were mounted after 35 days of culture on a materials testing machine (DMA Q800 Dynamic Mechanical Analyzer; TA Instruments, Newcastle, DE) in a radially unconfined stress relaxation test with the disks between impermeable platens, and hydrated in 0.9% saline containing protease inhibitors (Complete; Sigma). The secant modulus is related to the interaction between the solid and the liquid phase and is therefore an indication for the ability to retain water. The modulus measured at equilibrium depends strongly on the compressive stiffness of the (cartilaginous) solid matrix (29). Beads were rinsed 3 times in phosphate buffered saline and fixed for 2 hours at room temperature in 0.1M sodium cacodylate–buffered 1.5% glutaraldehyde (EM grade; Sigma) and 1% paraformaldehyde, pH 6.7, then rinsed 3 times in 0.15M sodium cacodylate. After fixation for 2 hours in 0.1M sodium cacodylate–buffered 1% osmium tetroxide, pH 6.7, the beads were dehydrated in a series of graded acetone and embedded in LX-112 Epon. Ultrathin sections (Leica Ultracut UCT; Leica Microsystems, Rijswijk, The Netherlands) were mounted on copper grids (300 mesh), contrasted with 2% uranyl acetate (10 minutes at 45°C) and lead citrate, and examined with a Zeiss 902 electron microscope (Carl Zeiss Instruments, Oberkochen, Germany). Images were obtained at 18,000× and 89,000× magnification. Four images per sample (89,000× magnification) were used to measure the width of as many collagen fibrils as possible in the cell-associated matrix with ImageJ 1.40g software (National Institutes of Health, Bethesda, MD). This resulted in the measurement of 38 fibrils in WT, 23 fibrils in double-deficient, 19 fibrils in type IX collagen–deficient, and 42 fibrils in COMP-deficient cultures. Each alginate culture experiment was repeated 2 times, each time using at least 4 pooled cartilage donors. Each experiment consisted of 3 × 7 beads per experimental condition for biochemical analyses, 3 beads for electron microscopic analysis, and 6 disks for mechanical testing. Statistical analysis was performed using GraphPad Prism 5.01 software (GraphPad Software, San Diego, CA). All data are presented as the mean ± SD. Samples from the WT and double-deficient groups were compared with a Kruskal-Wallis test followed by Dunn\'s post hoc multiple comparison tests. Immunofluorescence microscopy of the rib and sternum sections revealed the deposition of type IX collagen and COMP in WT mouse rib cartilage, while the lack of staining in double-deficient mice confirmed the absence of both proteins (Figures 1A–D). The staining for COMP and type IX collagen overlapped in ribs, but type IX collagen showed a broader expression pattern in sternal segments (Figures 1A and C). Antibodies directed against type II collagen stained all cartilaginous areas in both control and knockout mice, suggesting that type II collagen deposition is not substantially influenced by the absence of type IX collagen and COMP (Figures 1E and F). In contrast, matrilin 3 showed a partially changed localization. In WT mice, the staining pattern for matrilin 3 was similar to that for COMP, with matrilin 3 being expressed throughout the ribs and in the matrix of proliferating and differentiating chondrocytes (Figure 1G). When type IX collagen and COMP were missing, matrilin 3 staining was still detected in calcified cartilage but shifted toward the zone of resting chondrocytes located between adjacent sternal growth plates, where it normally is not detected. In addition, the matrilin 3 signal in the ribs was completely lost (Figure 1H). Staining with Alcian blue, a dye that binds to negatively charged glycosaminoglycans and thus to the major cartilage proteoglycan aggrecan, was not altered between genotypes and revealed no obvious malformations (Figures 1I and J). Immunofluorescence microscopy of rib cage cartilage. Sections obtained through the rib cage of wild-type (WT) and type IX collagen/cartilage oligomeric matrix protein (COMP)–double-deficient (dko) mice were incubated with antibodies directed against type IX collagen (A and B), COMP (C and D), type II collagen (E and F), and matrilin 3 (G and H). Sections were stained with hematoxylin and eosin (HE) and Alcian blue (I and J). Bar = 100 μm. The amount and solubility of matrilin 3 were examined by sequentially extracting cartilage. In tissue from WT mice, matrilin 3 was extracted with buffers containing only EDTA (buffer II) or the chaotropic agent GuHCl (buffer III), whereas in double-deficient mice some protein could be extracted with Tris buffered saline alone (buffer I). In addition to the increased solubility, the total amount of matrilin 3 that could be extracted was reduced when both type IX collagen and COMP were ablated (Figure 2A). This, together with the results from immunohistochemical analysis (Figure 1H), points to a decrease in the pool of matrilin 3 anchored in the matrix. Solubility of matrilin 3 and proteoglycans derived from rib cartilage. Matrilin 3 and proteoglycans were sequentially extracted in Tris buffered saline (TBS) alone (buffer I), TBS containing 10 mM EDTA (buffer II), or 4M GuHCl (buffer III). A, Immunoblots were used to detect matrilin 3, with ponceau staining as a loading control. B, Glycosaminoglycan (GAG) concentrations were determined in extracts obtained with the different buffers, from 1 mg wet weight of tissue. WT = wild-type; dko = type IX collagen/cartilage oligomeric matrix protein double-deficient. We also analyzed cartilage proteoglycans by the same extraction approach. Neither the solubility nor the total amount of extracted proteoglycans differed significantly between genotypes (Figure 2B). Chondrocytes were isolated from newborn WT and double-deficient mice and cultured in monolayer for 5 days. In cultures of WT cells, large quantities of both type IX collagen and COMP were integrated into an ECM, forming networks surrounding the chondrocytes (Figures 3A and C). A similar staining pattern was obtained for type II collagen, revealing deposition throughout the chondrocyte pericellular matrix regardless of the genotype (Figures 3E and F). Interestingly, matrilin 3 was completely lost from extracellular fibrillar structures when these lacked type IX collagen and COMP. However, matrilin 3 was still secreted and, in these cultures, nonspecifically coated the surface of the culture dish. Immunofluorescence microscopy of monolayer cultures of chondrocytes. Primary chondrocytes isolated from the rib cartilage of wild-type and double-deficient mice were cultured and stained with antibodies directed against type IX collagen (A and B), cartilage oligomeric matrix protein (COMP) (C and D), type II collagen (E and F), and matrilin 3 (G and H). Bar = 20 μm. See Figure 2 for other definitions. We determined the amount of proteoglycans deposited in the ECM after up to 7 days. Alcian blue binding assays revealed no significant difference between double-deficient and control chondrocytes in monolayer cultures at these early time points (results not shown). Protein and proteoglycan deposition in the ECM of long-term chondrocyte cultures in alginate. To analyze the involvement of type IX collagen and COMP in longer-term matrix assembly and maintenance, and to allow the quantitative analysis of different matrix compartments, isolated chondrocytes were cultured in alginate beads. Under these conditions, the chondrocytes synthesized type II collagen and not type I collagen during the 3-week period studied (results not shown). Western blot analyses of cell-associated matrix and further-removed matrix from both WT and double-deficient chondrocyte cultures showed less matrilin 3 deposition in the matrix of deficient cells (Figure 4A). Instead, more matrilin 3 was detected in the culture medium of these cells than in that of WT controls. A and B, Amount and compartmental distribution of matrilin 3 and proteoglycans, respectively, after 3 weeks of chondrocyte culture in alginate beads. A, Immunoblot analysis of cell-associated matrix (CM), further-removed matrix (FRM), and culture medium using antibodies directed against matrilin 3. Ponceau staining was used as a loading control. B, GAG distribution between the CM and the FRM. C, GAG distribution in the culture medium. Values in B and C are the mean ± SD (n = 6 samples comprising at least 3 alginate beads each). ∗︁ = P 0.05 versus WT. See Figure 2 for other definitions. Wild-type chondrocytes produced large amounts of proteoglycans over the 3-week period, resulting in proteoglycan deposition in the alginate bead of 10.2 ± 2.1 μg/bead (mean ± SD). In cultures of double-deficient chondrocytes, significantly fewer proteoglycans (mean ± SD 6.8 ± 2.1 μg/bead) were deposited. However, the relative distribution between cell-associated matrix and further-removed matrix was not significantly different when compared with WT cultures (Figure 4B). We also analyzed the culture medium harvested during the experiment. More proteoglycans were released into the culture medium of deficient chondrocytes (mean ± SD 3.0 ± 0.2 μg/bead; 30% of the total proteoglycan production) in comparison with WT cells (1.5 ± 0.1 μg/bead; 13% of the total proteoglycan production), indicating less retention of proteoglycans in a type IX collagen/COMP–deficient matrix (Figure 4C). The total collagen deposition was not significantly different in beads containing WT chondrocytes and those containing double-deficient chondrocytes (mean ± SD 6.3 ± 1.9 μg/bead and 5.6 ± 2.1 μg/bead, respectively). Interestingly, the relative distribution within the alginate bead was altered, with 23% of the total collagen being deposited in the further-removed matrix of chondrocytes isolated from double-deficient mice and 12% in the further-removed matrix of WT chondrocytes (Figure 5A). Less collagen was observed in culture medium collected during the experiment with double-deficient chondrocytes (mean ± SD 4.0 ± 0.3 μg/bead) than in the medium of the WT cells (5.26 ± 0.7 μg/bead) (Figure 5B). This difference in absolute release did not result in a significant relative difference when compared with total collagen production. Collagen was less crosslinked when type IX collagen and COMP were absent, as determined by measuring the number of hydroxypyridinoline crosslinks per collagen triple helix (Figure 5C). Distribution of collagen, collagen crosslinks, and matrix metalloproteinase (MMP) activity after 3 weeks of chondrocyte culture in alginate beads. A and B, Collagen distribution between the cell-associated matrix (CM) and the further-removed matrix (FRM) (A) and in the culture medium (B). Values are the mean ± SD (n = 6). # = significant difference (P 0.05) in distribution; ∗︁ = P 0.05 versus WT. C, Hydroxylysylpyridinoline (HP) crosslinks in newly formed collagen. Values are the mean ± SD (n = 4). ∗︁ = P 0.01 versus WT. D, Overall MMP activity as measured in the culture medium, using a fluorescent substrate. Values are the mean ± SD relative fluorescence units (RFUs) per second (n = 4). See Figure 2 for other definitions. A decreased amount of collagen in the medium can result from less synthesis or less degradation. MMPs are major matrix-degrading enzymes that influence matrix quality and quantity. We determined the overall MMP activity in the culture media and observed comparable activity irrespective of the genotype (Figure 5D). The secant modulus and the equilibrium aggregate modulus were examined to test whether changes in the amount and distribution of produced ECM components affect mechanical properties of the alginate beads containing chondrocytes. The peak force and eventually the ability to retain water, described by the secant modulus, were not altered in 3-week cultures of chondrocytes lacking type IX collagen and COMP when compared with WT chondrocytes (mean ± SD 5,070 ± 1,623 Pa for WT and 4,588 ± 890 Pa for double-deficient cultures). Also, stiffness, indicated by the equilibrium aggregate modulus, did not differ significantly between genotypes (767 ± 232 Pa for WT and 970 ± 283 Pa for double-deficient cultures). After 3 weeks of culture, complete alginate beads were processed for electron microscopy. The matrix deposited by WT and type IX collagen/COMP–double-deficient chondrocytes appeared similar, with some collagen fibrils positioned parallel to the cell surface and the fibrils being relatively closely spaced (Figures 6A and D). In cultures of type IX collagen/COMP–double-deficient chondrocytes, the fibril diameter was comparable with that observed in cultures of WT cells (Figure 6E). Electron microscopy of chondrocytes and collagen fibrils in alginate beads cultured for 3 weeks. A–D, Alginate beads containing wild-type (WT) (A), type IX collagen–single-knockout (B), cartilage oligomeric matrix protein (COMP)–deficient (C), or type IX collagen/COMP double-deficient (dko) (D) mouse chondrocytes were submitted to electron microscopy. Bars = 2 μm. E, Diameters of individual collagen fibrils formed by WT, type IX collagen–deficient, COMP-deficient, or type IX collagen/COMP–double-deficient mouse chondrocytes were measured. Bars show the mean ± SD. ∗︁ = P 0.001. We were surprised that neither the appearance nor the diameter of fibrils produced by double-deficient cells differed compared with WT cultures, because we previously detected an increased collagen fibril diameter in cartilage from type IX collagen–single-knockout mice (7). We therefore analyzed the matrix produced by chondrocytes lacking expression of only type IX collagen or COMP in alginate beads, under identical conditions. In agreement with earlier studies on intact cartilage, electron microscopy of type IX collagen–deficient cultures (Figure 6B) showed a less organized matrix and a significantly larger fibril diameter (P 0.001) than that in either WT or type IX collagen/COMP–double-deficient cultures (Figure 6E). Collagen fibrils formed in the COMP-deficient culture (Figure 6C) had no altered fibril diameter compared with the WT culture (Figure 6E). Even though the exact functions of collagen fibril–associated proteins, such as type IX collagen, COMP, and matrilin, are far from clear, as a group these proteins are thought to be critically important for skeletal development and presumably for the development of musculoskeletal diseases. Mice lacking type IX collagen have an increased collagen fibril diameter and a loss of matrilin 3 anchorage (7), whereas COMP-deficient mice have no similar defects in matrix assembly or fibril diameter (11). Until now, it has been unclear whether the loss of both type IX collagen and COMP enhances the matrix assembly phenotype observed in type IX collagen–deficient mice, and whether type IX collagen and COMP play compensatory or antagonistic roles during matrix assembly. First, we addressed the anchorage of matrix proteins in vivo. Immunofluorescence staining confirmed the absence of type IX collagen and COMP in double-deficient cartilage. Type II collagen deposition was not visibly altered in the rib cages of double-deficient mice. In contrast, matrilin 3 was lost from most of the cartilaginous tissues of double-deficient mice, similar to what was previously reported for mice deficient in only type IX collagen (7). Retention of matrilin 3 was observed in the zone between adjacent sternal growth plates as well as in calcified cartilage, indicating the presence of new interaction partners within these regions of type IX collagen/COMP–deficient tissue. In agreement with the lost matrilin 3 staining, the protein was more easily extracted from the tissue. Differences in the deposition of aggrecan were not detected by glycosaminoglycan analysis of mouse cartilage extracts, and histologic sections obtained through the rib cages of WT and double-deficient mice revealed no obvious malformations. This is different from the tibial growth plate cartilage, where hypocellular areas and impaired chondrocyte alignment were observed (17). Apparently, different types of cartilage have different requirements for collagen fibril–associated proteins. To analyze matrix assembly and protein deposition in greater detail, we employed both short-term and long-term cell culture systems. In short-term chondrocyte cultures, matrilin 3 deposition was clearly influenced by the loss of type IX collagen and COMP. In earlier studies employing type IX collagen–deficient cells, matrilin 3 was secreted into the medium instead of being incorporated into the matrix (7). However, if COMP also was ablated, amorphous matrilin 3 aggregates bound to the surface of the culture dish. A strong interaction between matrilin 3 and COMP has been described (30). Although matrilins show poor solubility, COMP is highly hydrophilic. It is likely that complex formation between matrilin 3 and COMP increases matrilin 3 solubility in the cell culture supernatant and prevents deposition on the surfaces. In the tibiae of type IX collagen/COMP–deficient mice (17), matrilin 3 was amorphously deposited in the central region of the epiphyseal cartilage, a phenomenon that was not seen when only type IX collagen was lacking. This indicates that COMP may also act as a carrier for matrilin 3 in vivo. Intense type II collagen staining was detected in the matrix of both WT and double-deficient chondrocytes, showing the assembly of pericellular collagen fibrils regardless of genotype. Matrilin 3 binds with low affinity directly to type II collagen in vitro (30), but apparently type II collagen alone cannot provide adequate tissue anchorage for matrilin 3 if type IX collagen and COMP are missing. In agreement with the unchanged in vivo proteoglycan solubility, the amount deposited in monolayer culture did not differ between WT and double-deficient cells. In monolayer cultures, the amount of matrix formed is limited, and differences in aggrecan retention within this matrix may be difficult to detect. We also used a 3-D cell culture model. This culture system can be used for longer-term experiments, because it ensures phenotypic stability of chondrocytes (31-33). Again, matrilin 3 was less well anchored in the matrix produced by double-deficient cells and was instead released into the supernatant. Similarly, fewer proteoglycans were present within the matrix. Because COMP can bind to aggrecan (34), the main aggregate-forming proteoglycan in cartilage, the loss of COMP might result in decreased aggrecan anchorage, an effect that could be enhanced by the secondary loss of matrilin 3, because matrilins may link aggrecan to microfibrillar networks (35). This effect was not detected in vivo, probably due to the high density of collagen fibrils that limits aggrecan diffusion and thereby compensates for the decreased matrix anchorage. The matrix assembled in alginate beads appears less dense and allows the detection of differences in proteoglycan retention. Matrilin 3 is smaller and can diffuse out of both types of matrix if anchorage to type IX collagen and COMP is lost. The distribution of proteoglycans between pericellular and interterritorial matrix was not altered between genotypes, and the total amount of collagen, mainly type II collagen (32), in the beads did not differ significantly. However, the deposition of collagen was shifted from the cell-associated matrix toward the further-removed matrix, presumably due to better diffusion of collagen through the alginate bead. This increase in diffusion might be a direct consequence of less collagen crosslinking. Accordingly, the ratio between aggrecan and collagen decreases, particularly in the interterritorial matrix. These clear differences in matrix organization did not lead to detectable changes in mechanical properties. Despite the compositional differences in cartilage matrix formed in the absence of both type IX collagen and COMP, electron microscopy revealed no obvious changes in the diameter of the collagen fibrils formed. This was surprising, because an increased fibril diameter was observed in mice lacking only type IX collagen (7). We also observed this increase in the matrix of type IX collagen–deficient chondrocytes cultured in alginate. Type IX collagen is thought to limit lateral fibril growth by binding to the surface of the growing fibril (6). Alternatively, COMP has been shown to act as a catalyst of in vitro collagen fibrillogenesis, and fibrils formed in the absence of COMP had a more heterogeneous diameter than those formed in its presence (10). Results from a study of equine tendon led to the suggestion that COMP may also have a function in modulating fibril growth in vivo (36). However, when comparing the collagen fibril diameter in WT and COMP-deficient mice, no differences were detected (11). Our results using COMP-deficient chondrocyte cultures confirm this in vivo observation, although we observed a tendency toward smaller fibrils in our experiments. Surprisingly, in the absence of both type IX collagen and COMP, fibrils of normal diameter were formed, even though the lack of type IX collagen alone resulted in a significantly increased fibril diameter. This means that the dramatic increase in fibril diameter in type IX collagen–deficient cultures was reversed when COMP was also absent, suggesting that type IX collagen limits the fibril diameter, while COMP might weakly promote lateral growth. The antagonistic effect of type IX collagen and COMP in fibril assembly implies that the relative abundance of these proteins may regulate collagen fibril diameter in vivo. It is possible that disturbances in the secretion and/or function of type IX collagen and COMP, as seen in human multiple epiphyseal dysplasia or pseudoachondroplasia, can affect collagen fibril assembly and, through the resulting matrix reorganization, promote the development of OA that is connected with these disorders. The synthesis, degradation, and assembly of ECM components can be influenced by growth factors and cytokines that are known to be produced in OA joints, such as transforming growth factor β (22, 37). Although type II collagen has been most extensively studied in this regard, an increasing body of work now points to an effect of these cytokines on COMP production and degradation (38-40). It is less clear how growth factors affect type IX collagen expression. The observed changes in the deposition of matrix proteins caused by the loss of type IX collagen and COMP point to a regulatory influence on cartilage matrix assembly and may thereby influence the susceptibility for cartilage degeneration. We are grateful to Wendy Koevoet for technical assistance with the alginate cultures, and to the Department of Pathology, Erasmus Medical Center, University Medical Center Rotterdam, for electron microscopy. 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